Bundle sheath chloroplasts have been isolated from Zea mays leaves by a procedure involving enzymic digestion of mechanically prepared strands of bundle sheath cells followed by gentle breakage and filtration.The resulting crude chloroplast preparation was enriched by Percoll density layer centrifugation to yield intact chloroplasts (about 20 micrograms chlorophyll per 10-gram leaf tissue) with high metabolic activities. Based on activities of marker enzymes in the chloroplast and bundle sheath cell extracts, the chloroplasts were essentially free of contamination by other organelles and cytoplasmic material, and were generally about 70% intact. Chlorophyll a/b ratios were high (about 10 Bundle sheath cells of C4 plants are the site of C4 acid decarboxylation and refixation ofthe released CO2 via the PCR' cycle. In C4 species of the NADP-ME type malate decarboxylation is thought to occur in the chloroplasts via the action of NADP-ME (8,15,27). This enzyme generates a portion of the NADPH required for photosynthetic CO2 fixation, so that malate decarboxylation is considered to be closely linked to the PCR cycle (5, 26). The evidence for these functions is largely based on enzyme localization studies using chloroplasts isolated from maize by nonaqueous techniques (30), more recently from bundle sheath protoplasts ofFlaveria trinervia (27), and from studies 'Abbreviations: PCR, photosynthetic carbon reduction; PGA, 3-phosphoglyceric acid; PEP, phosphoenolpyruvate; RuBP, ribulose-l1,5-bisphosphate; Ru5P, ribulose-5-phosphate; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; R5P, ribose-5-phosphate; ME, malic enzyme. ofthe metabolic activities of isolated bundle sheath cells ofmaize (5,7,12,14) and other C4 species of the NADP-ME type (6, 12, 26, 29). Other than one brief report (29) it has not been possible to confirm these proposed metabolic activities using functional bundle sheath chloroplasts isolated from NADP-ME type C4 species due to the difficulty in preparing these organelles (see Edwards and Huber [8]).Rathnam and Edwards (28) reported on the preparation and enzyme content of bundle sheath chloroplasts from all three C4 subgroups. Metabolic activities were also presented (29). However, the preparation method used has not been repeated elsewhere and was ineffective in our hands. Isolation ofmaize bundle sheath chloroplasts was also reported by Horvath et al. (17) but these chloroplasts displayed negligible CO2 assimilation.Chloroplast fragment preparations from C4 bundle sheath cells have been useful in studies of the Hill reaction, photosynthetic electron transport, and electron transport chain components (1,11,24,26). Bundle sheath chloroplasts capable of phenazine methosulfate-mediated cyclic photophosphorylation have also been prepared from maize (2). This work has indicated that the agranal bundle sheath chloroplasts of NADP-ME type C4 species are deficient in PSII and have higher Chl a/b ratios than chloroplasts from other plant sources. It appears likely that the resulting d...

The light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) was inhibited in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Decreases in the velocity and magnitude of light activation correlated with the overall reduction in CO2 fixation rates. Responses of osmotically stressed chloroplasts to both varying pH and exogeous dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerete (PGA) were examined. In the presence of DHAP, the absolute rate of CO2 fixation was increased and this increase was most pronounced at alkaline pH. Enhanced light activation of these enzymes was also observed under these conditions. However, in the presence of PGA, similar increases in photosynthetic rate and enzyme activation were not evident. Light-dependent stromal alkalization was unaffected by the stress treatments. Inhibition of light activation under hypertonic conditions is discussed in terms of substrate availability, possible alterations of the redox state of ferredoxin and associated electron carriers, and inhibited enzyme-enzyme or enzyme-substrate interactions involved in the light activation process.

Conditions for optimai CO2 fixation and malte decarboxylation by isolated bundle sheath chloroplasts from Zea may: were examined. The relative rates of these processes varied according to